Metabolism of tamoxifen by recombinant human cytochrome P450 enzymes: formation of the 4-hydroxy, 4'-hydroxy and N-desmethyl metabolites and isomerization of trans-4-hydroxytamoxifen.

The cytochrome P450 (P450)-mediated biotransformation of tamoxifen is important in determining both the clearance of the drug and its conversion to the active metabolite, trans-4-hydroxytamoxifen. Biotransformation by P450 forms expressed extrahepatically, such as in the breast and endometrium, may be particularly important in determining tissue-specific effects of tamoxifen. Moreover, tamoxifen may serve as a useful probe drug to examine the regioselectivity of different forms. Tamoxifen metabolism was investigated in vitro using recombinant human P450s. Forms CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7 were coexpressed in Escherichia coli with recombinant human NADPH-cytochrome P450 reductase. Bacterial membranes were harvested and incubated with tamoxifen or trans-4-hydroxytamoxifen under conditions supporting P450-mediated catalysis. CYP2D6 was the major catalyst of 4-hydroxylation at low tamoxifen concentrations (170 +/- 20 pmol/40 min/0.2 nmol P450 using 18 microM tamoxifen), but CYP2B6 showed significant activity at high substrate concentrations (28.1 +/- 0.8 and 3.1 +/- 0.5 nmol/120 min/0.2 nmol P450 for CYP2D6 and CYP2B6, respectively, using 250 microM tamoxifen). These two forms also catalyzed 4'-hydroxylation (13.0 +/- 1.9 and 1.4 +/- 0.1 nmol/120 min/0.2 nmol P450, respectively, for CYP2B6 and CYP2D6 at 250 microM tamoxifen; 0.51 +/- 0.08 pmol/40 min/0.2 nmol P450 for CYP2B6 at 18 microM tamoxifen). Tamoxifen N-demethylation was mediated by CYP2D6, 1A1, 1A2, and 3A4, at low substrate concentrations, with contributions by CYP1B1, 2C9, 2C19 and 3A5 at high concentrations. CYP1B1 was the principal catalyst of 4-hydroxytamoxifen trans-cis isomerization but CYP2B6 and CYP2C19 also contributed.     

Alpha-hydroxylation of tamoxifen and toremifene by human and rat cytochrome P450 3A subfamily enzymes

An increased risk of developing endometrial cancer is observed in breast cancer patients treated with tamoxifen (TAM) and in healthy women undergoing TAM chemoprevention therapy. TAM-DNA adducts were detected in the endometrium of women taking TAM (Shibutani, S., et al. (2000) Carcinogenesis 21, 1461-1467) and are formed primarily through O-sulfonation of alpha-hydroxytamoxifen (alpha-OHTAM). To explore the genotoxicic mechanisms of TAM, TAM was incubated with one of multiple human cytochrome P450 enzymes, i.e., P450 1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, 3A7, 4A11, 4F2, 4F3A, or 4F3B, in a NADPH regenerating system, and the metabolites were identified using HPLC/UV analysis with authentic standards. Among the 18 human P450 enzymes, P450 3A4 generated a significant amount of alpha-OHTAM. When some rat P450 enzymes were examined, P450 3A2 also catalyzed alpha-hydroxylation of TAM. Similarly, human P450 3A4 and rat P450 3A1 and 3A2 converted toremifene (TOR, a chlorinated TAM analogue) to alpha-hydroxytoremifene (alpha-OHTOR). The formation of alpha-OHTAM and alpha-OHTOR by these P450 enzymes was confirmed by tandem mass spectroscopy. Only the P450 3A subfamily enzymes are able to alpha-hydroxylate TAM and TOR. Although the formation of alpha-OHTOR by these enzymes was much higher than that of alpha-OHTAM, TOR is known to be much less genotoxic than TAM. The results support our proposed mechanism that the lower genotoxicity of TOR is due to limited O-sulfonation of alpha-OHTOR by hydroxysteroid sulfotransferases, resulting in the poor formation of DNA adducts (Shibutani, S., et al. (2001) Cancer Res. 61, 3925-3931).     

Potential Beneficial Metabolic Interactions Between Tamoxifen and Isoflavones via Cytochrome P450-mediated Pathways in Female Rat Liver Microsomes

PURPOSE: This study aims to evaluate a cytochrome P450-based tamoxifen-isoflavone interaction and to determine the mechanisms responsible for inhibitory effects of isoflavones (e.g., genistein) on the formation of alpha-hydroxytamoxifen. METHODS: Metabolism studies were performed in vitro using female rat liver microsomes. The effects of genistein and an isoflavone mixture on tamoxifen metabolism and the inhibition mechanism were determined using standard kinetic analysis, preincubation, and selective chemical inhibitors of P450. RESULTS: Metabolism of tamoxifen was saturable with Km values of 4.9+/-0.6, 14.6+/-2.2, 25+/-5.9 microM and Vmax values of 34.7+/-1.4, 297.5+/-19.2, 1867+/-231 pmol min(-1) mg(-1) for a-hydroxylation, N-desmethylation, and N-oxidation, respectively. Genistein (25 microM) inhibited alpha-hydroxylation at 2.5 microM tamoxifen by 64% (p < 0.001) but did not affect the 4-hydroxylation, N-desmethylation, and N-oxidation. A combination of three (genistein, daidzein, and glycitein) to five isoflavones (plus biochanin A and formononetin) inhibited tamoxifen alpha-hydroxylation to a greater extent but did not decrease the formation of identified metabolites. The inhibition on alpha-hydroxylation by genistein was mixed-typed with a Ki, value of 10.6 microM. Studies using selective chemical inhibitors showed that tamoxifen alpha-hydroxylation was mainly mediated by rat CYP1A2 and CYP3A1/2 and that genistein 3'-hydroxylation was mainly mediated by rat CYP1A2, CYP2C6 and CYP2D1. CONCLUSIONS: Genistein and its isoflavone analogs have the potential to decrease side effects of tamoxifen through metabolic interactions that inhibit the formation of a-hydroxytamoxifen via inhibition of CYP1A2.     

Bioactivation of tamoxifen by recombinant human cytochrome p450 enzymes

Tamoxifen is a major drug used for adjuvant chemotherapy of breast cancer; however, its use has been associated with a small but significant increase in risk of endometrial cancer. In rats, tamoxifen is a hepatocarcinogen, and DNA adducts have been observed in both rat and human tissues. Tamoxifen has been shown previously to be metabolized to reactive products that have the potential to form protein and DNA adducts. Previous studies have suggested a role for P450 3A4 in protein adduct formation in human liver microsomes, via a catechol intermediate; however, no clear correlation was seen between P450 3A4 content of human liver microsomes and adduct formation. In the present study, we investigated the P450 forms responsible for covalent drug-protein adduct formation and the possibility that covalent adduct formation might occur via alternative pathways to catechol formation. Recombinant P450 3A4 catalyzed adduct formation, and this correlated with the level of uncoupling in the P450 incubation, consistent with a role of reactive oxygen species in potentiating adduct formation after enzymatic formation of the catechol metabolite. Whereas P450s 1A1, 2D6, and 3A5 generated catechol metabolite, no covalent adduct formation was observed with these forms. By contrast, P450 2B6, 2C19, and rat liver microsomes catalyzed drug-protein adduct formation but not catechol formation. Drug protein adducts formed specifically with P450 3A4 in incubations using membranes isolated from bacteria expressing P450 3A4 and reductase, as well as in reconstitutions of purified 3A4, suggesting that the electrophilic species reacted preferentially with the P450 enzymes concerned.     

Synthesis and reactivity of a potential carcinogenic metabolite of tamoxifen: 3,4-dihydroxytamoxifen-o-quinone

Although tamoxifen is approved for the treatment of hormone-dependent breast cancer as well as for the prevention of breast cancer in high-risk women, several studies in animal models have shown that tamoxifen is heptocarcinogenic, and in humans, tamoxifen has been associated with an increased risk of endometrial cancer. One potential mechanism of tamoxifen carcinogenesis could involve metabolism of tamoxifen to 3,4-dihydroxytamoxifen followed by oxidation to a highly reactive o-quinone which has the potential to alkylate and/or oxidize cellular macromolecules in vivo. In the study presented here, we synthesized the 3,4-dihydroxytamoxifen, prepared its o-quinone chemically and enzymatically, and studied the reactivity of the o-quinone with GSH and deoxynucleosides. The E (trans) and Z (cis) isomers of 3,4-dihydroxytamoxifen were synthesized using a concise synthetic pathway (four steps). This approach is based on the McMurry reaction between the key 4-(2-chloroethoxy)-3,4-methylenedioxybenzophenone and propiophenone, followed by selective removal of the methylenedioxy ring of (E, Z)-1-[4-[2-(N,N-dimethylamino)ethoxy]phenyl]-1-(3, 4-methylenedioxyphenyl)-2-phenyl-1-butene with BCl(3). Oxidation of 3,4-dihydroxytamoxifen by activated silver oxide or tyrosinase gave 3,4-dihydroxytamoxifen-o-quinone as a mixture of E and Z isomers. The resulting o-quinone has a half-life of approximately 80 min under physiological conditions. Reaction of the o-quinone with GSH gave two di-GSH conjugates and three mono GSH conjugates. Incubation of 3,4-dihydroxytamoxifen with GSH in the presence of microsomal P450 gave the same GSH conjugates which were also detected in incubations with human breast cancer cells (MCF-7). Reaction of 3, 4-dihydroxytamoxifen-o-quinone with deoxynucleosides gave only thymidine and deoxyguanosine adducts; neither deoxyadenosine nor deoxycytosine adducts were detected. Preliminary studies conducted with human breast cancer cell lines showed that 3, 4-dihydroxytamoxifen exhibited cytotoxic potency similar to that of 4-hydroxytamoxifen and tamoxifen in an estrogen receptor negative (ER(-)) cell line (MDA-MB-231); however, in the ER(+) cell line (MCF-7), the catechol metabolite was about half as toxic as the other two compounds. Finally, in the presence of microsomes and GSH, 4-hydroxytamoxifen gave predominantly quinone methide GSH conjugates as reported in the previous paper in this issue [Fan, P. W., et al. (2000) Chem. Res. Toxicol. 13, XX-XX]. However, in the presence of tyrosinase and GSH, 4-hydroxytamoxifen was primarily converted to o-quinone GSH conjugates. These results suggest that the catechol metabolite of tamoxifen has the potential to cause cytotoxicity in vivo through formation of 3,4-dihydroxytamoxifen-o-quinone.     

Cytochrome P450-mediated metabolism of haloperidol and reduced haloperidol to pyridinium metabolites

Haloperidol (HP) has been reported to undergo cytochrome P450 (P450)-mediated metabolism to potentially neurotoxic pyridinium metabolites; however, the chemical pathways and specific enzymes involved in these reactions remain to be identified. The aims of the current study were to (i) fully identify the cytochrome P450 enzymes capable of metabolizing HP to the pyridinium metabolite, 4-(4-chlorophenyl)-1-(4-fluorophenyl)-4-oxobutylpyridinium (HPP(+)), and reduced HP (RHP) to 4-(4-chlorophenyl)-1-(4-fluorophenyl)-4-hydroxybutylpyridinium (RHPP(+)); and (ii) determine whether 4-(4-chlorophenyl)-1-(4-fluorophenyl)-4-oxobutyl-1,2,3,6-tetrahydropyridine (HPTP) and 4-(4-chlorophenyl)-1-(4-fluorophenyl)-4-hydroxybutyl-1,2,3,6-tetrahydropyridine (RHPTP) were metabolic intermediates in these pathways. In vitro studies were conducted using human liver microsomal preparations and recombinant human cytochrome P450 enzymes (P450s 1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19 2D6, 2E1, 3A4, 3A5, and 3A7) expressed in bicistronic format with human NADPH cytochrome P450 reductase in Escherichia coli membranes. Pyridinium formation from HP and RHP was highly correlated across liver preparations, suggesting the same enzyme or enzymes were responsible for both reactions. Cytochrome P450s 3A4, 3A5, and 3A7 were the only recombinant enzymes which demonstrated significant catalytic activity under optimized conditions, although trace levels of activity could be catalyzed by NADPH-P450 reductase alone. NADPH-P450 reductase-mediated activity was inhibited by reduced glutathione but not catalase or superoxide dismutase, suggesting O(2)-dependent oxidation. No evidence was obtained to support the contention that HPTP and RHPTP are intermediates in these pathways. K(m) values for HPP(+) (34 +/- 5 microM) and RHPP(+) (64 +/- 4 microM) formation by recombinant P450 3A4 agreed well with those obtained with human liver microsomes, consistent with P450 3A4 being the major catalyst of pyridinium metabolite formation in human liver.     

Mechanism-based inactivation of cytochrome P450 3A4 by 4-ipomeanol

Earlier phase I and II clinical studies showed that 4-ipomeanol produced selective hepatotoxicity. To investigate the mechanism of bioactivation of 4-ipomeanol, we thoroughly studied the interaction of 4-ipomeanol with human cytochrome P450 3A4 (EC 1.14.14.1). 4-Ipomeanol produced a time- and concentration-dependent inactivation of P450 3A4. More than 80% of the P450 3A4 activity was lost after its incubation with 4-ipomeanol at the concentration of 75 microM in 12 min. The inactivation was characterized by a rate of inactivation (kinact) of 0.15 min(-1) and by an inactivation potency (KI) of 20 microM. In addition, the inhibition of P450 3A4 by 4-ipomeanol was NADPH-dependent and irreversible. Glutathione, catalase, and superoxide dismutase failed to protect P450 3A4 from inactivation by 4-ipomeanol. The presence of testosterone, a substrate of P450 3A4, protected the enzyme from inactivation. The estimated partition ratio of the inactivation was approximately 257. Covalent binding studies demonstrated that reactive metabolites of 4-ipomeanol modified P450 3A4 but not P450 reductase (EC 1.6.2.4). The stoichiometry of binding between reactive metabolites of radiolabeled 4-ipomeanol and P450 3A4 was approximately 1.5:1. In addition to P450 3A4, reactive metabolites of 4-ipomeanol were found to covalently bind to other proteins. 4-Ipomeanol failed to inactivate P450 1A2 in human liver microsomes. In conclusion, 4-ipomeanol irreversibly inhibited P450 3A4, and it was characterized as a mechanism-based inactivator of P450 3A4. This finding facilitates the understanding of the mechanism of bioactivation of 4-ipomeanol by human hepatic enzymes.     

Identification of cytochrome P450 enzymes involved in the metabolism of zotepine, an antipsychotic drug, in human liver microsomes

1. Studies using human liver microsomes and recombinant human cytochrome P450 (P450) enzymes and flavin-containing monooxygenase (FMO) were performed to identify the enzymes responsible for the formation of zotepine metabolites in man. 2. Human liver microsomes produced four metabolites and a tentative order of importance was: norzotepine, 3-hydroxyzotepine, zotepine S-oxide and 2-hydroxyzotepine. Zotepine N-oxide was also detected, but it could not be quantified. 3. The rates of formation of the major metabolite, norzotepine, and zotepine S-oxide (at a substrate concentration of 20 microM) were significantly correlated with the testosterone 6beta-hydroxylase activities and CYP3A4 contents of the 12 different human liver microsomal samples. Inhibition studies with P450 enzyme selective inhibitors and anti-rat CYP3A2 antibodies also indicated a predominant role of CYP3A4 in the formation of norzotepine and zotepine S-oxide. Furafylline and sulphaphenazole inhibited the N-demethylation of zotepine by up to approximately 30%. 4. Correlation and inhibition data for the 2- and 3-hydroxylation of zotepine were consistent with the predominant role of CYP1A2 and 2D6 in the formation of these metabolites, respectively. 5. Recombinant CYP1A1, 1A2, 2B6, 2C19, 3A4 and 3A5 efficiently catalysed N-demethylation of zotepine. CYP1A1, 1A2, 2B6 and 3A4 were also active for S-oxidation. CYP1A2 and 2D6*1-Val374 efficiently produced 2-hydroxyzotepine and 3-hydroxyzotepine, respectively. Recombinant human FMO3 did not catalyse zotepine S-oxidation. 6. These results suggest that both the N-demethylation and S-oxidation of zotepine are mediated mainly by CYP3A4, and that CYP1A2 and 2D6 play an important role in the 2- and 3-hydroxylation of zotepine, respectively.     

Formation of tamoxifen-DNA adducts in human endometrial explants exposed to alpha-hydroxytamoxifen

An increased risk of developing endometrial cancer has been observed in women receiving tamoxifen (TAM) endocrine therapy and chemoprevention. The genotoxic damage induced by TAM metabolites may be involved in the development of endometrial cancer. To investigate the capability of endometrial tissues to form TAM-DNA adducts, primary cultured human endometrial explants were exposed to alpha-hydroxytamoxifen (alpha-OHTAM) and used for quantitative analysis of TAM-DNA adducts, using (32)P-postlabeling/HPLC analysis. A trans isoform of alpha-(N(2)-deoxyguanosinyl)tamoxifen (dG-N(2)-TAM) was detected as the major adduct in eight of nine endometrial explants exposed to 100 microM alpha-OHTAM at levels of 7.7 +/- 5.3 (mean +/- SD) adducts/10(7) nucleotides. Approximately 25- and 37-fold lower amounts of the cis form of dG-N(2)-TAM and another trans isoform were also detected. The dG-N(2)-TAM adduct (3.3 adducts/10(7) nucleotides) was detected in one of three endometrial explants exposed to 25 microM alpha-OHTAM. No TAM-DNA adducts were detected in any unexposed tissues. These results indicate that TAM-DNA adducts are capable of forming through O-sulfonation and/or O-acetylation of alpha-OHTAM in the endometrium. The endometrial explant culture can be used as a model system to explore the genotoxic mechanism of antiestrogens for humans.     

Roles of human hepatic cytochrome P450s 2C9 and 3A4 in the metabolic activation of diclofenac

Recently, it was shown that diclofenac was metabolized in rats to reactive benzoquinone imines via cytochrome P450-catalyzed oxidation. These metabolites also were detected in human hepatocyte cultures in the form of glutathione (GSH) adducts. This report describes the results of further studies aimed at characterizing the human hepatic P450-mediated bioactivation of diclofenac. The reactive metabolites formed in vitro were trapped by GSH and analyzed by LC/MS/MS. Thus, three GSH adducts, namely, 5-hydroxy-4-(glutathion-S-yl)diclofenac (M1), 4'-hydroxy-3'-(glutathion-S-yl)diclofenac (M2), and 5-hydroxy-6-(glutathion-S-yl)diclofenac (M3), were identified in incubations of diclofenac with human liver microsomes in the presence of NADPH and GSH. The formation of the adducts was taken to reflect the intermediacy of the corresponding putative benzoquinone imines. While M2 was the dominant metabolite over a substrate concentration range of 10-50 microM, M1 and M3 became equally important products at >/=100 microM diclofenac. The formation of M2 was inhibited by sulfaphenazole or an anti-P450 2C9 antibody (5-10% of control values). The formation of M1 and M3 was inhibited by troleandomycin, ketoconazole, or an anti-P450 3A4 antibody (30-50% of control values). In studies in which recombinant P450 isoforms were used, M2 was generated only by P450 2C9-catalyzed reaction, while M1 and M3 were produced by P450 3A4-catalyzed reaction. Good correlations were established between the extent of formation of M2 and P450 2C9 activities (r = 0.93, n = 10) and between the extent of formation of M1 and M3 and P450 3A4 activities (r = 0.98, n = 10) in human liver microsomal incubations. Taken together, the data suggest that the biotransformation of diclofenac to M2 is P450 2C9-dependent, whereas metabolism of the drug to M1 and M3 involves mainly P450 3A4. Although P450s 2C9 and 3A4 both catalyze the bioactivation of diclofenac, P450 2C9 is capable of producing the benzoquinone imine intermediate at lower drug concentrations which may be more clinically relevant.     

Reversible and irreversible inhibition of CYP3A enzymes by tamoxifen and metabolites

1. Preliminary studies have identified cytochrome P450 (CYP) 3A4 and CYP1B1 as the human CYPs inhibited by tamoxifen. To quantify the inhibitory potency of tamoxifen and its major metabolites, the metabolism of three substrates of CYP3A, midazolam, diltiazem and testosterone, and 7-ethoxyresorufin as a substrate of CYP1B1 were examined in catalytic assays carried out using human liver microsomes and cDNA-expression systems. 2. Tamoxifen, N-desmethyltamoxifen, 4-hydroxytamoxifen and 3-hydroxytamoxifen reversibly inhibited midazolam 1'-hydroxylation, diltiazem N-demethylation and testosterone 6beta-hydroxylation with K(i) ranging from 3 to 37 micro M in human liver microsomes. Tamoxifen, N-desmethyltamoxifen, 4-hydroxytamoxifen and 3-hydroxytamoxifen also reversibly inhibited the activity of cDNA-expressed CYP3A4, CYP3A5 and CYP1B1. 3. Tamoxifen and N-desmethyltamoxifen exhibited time-dependent inactivation of testosterone 6beta-hydroxylation by cDNA-expressed CYP3A4 (+ cytochrome b5) yielding k(inact) and K(i) of 0.04 min(-1) and 0.2 micro M for tamoxifen and 0.08 min(-1) and 2.6 micro M for N-desmethyltamoxifen. A metabolic intermediate complex (MIC) was also formed by tamoxifen and N-desmethyltamoxifen with CYP3A4 (+ cytochrome b5) and CYP3A4 but not with CYP3A5 or CYP3A7. Pre-incubation with 4-hydroxytamoxifen and 3-hydroxytamoxifen did not result in any CYP3A inactivation or detectable MIC formation. There was no detectable time-dependent inactivation or MIC formation with tamoxifen or metabolites with CYP1B1. 4. These data indicate that tamoxifen and its three major metabolites are effective inhibitors of CYP3A in vitro and that tamoxifen and N-desmethyltamoxifen are effective mechanism-based inhibitors. Thus, caution should be exercised when tamoxifen is coadministered with other CYP3A substrates.     

In vitro inhibitory effects of troglitazone and its metabolites on drug oxidation activities of human cytochrome P450 enzymes: comparison with pioglitazone and rosiglitazone

1. Investigated were the effects of a new oral antidiabetic drug, troglitazone, and its three metabolites and antidiabetic drug candidates pioglitazone and rosiglitazone on xenobiotic oxidations catalyzed by nine recombinant human cytochrome P450 (P450 or CYP) enzymes and by human liver microsomes. 2. Troglitazone (5 microM) significantly inhibited CYP2C8-dependent paclitaxel 6alpha-hydroxylation and CYP2C9-dependent S-warfarin 7-hydroxylation. On the other hand, pioglitazone and rosiglitazone (50 microM) only slightly inhibited these xenobiotic oxidation activities catalyzed by CYP2C enzymes. 3. The inhibitory potential of troglitazone (50% inhibition concentration, IC50) was approximately 5 microM for drug oxidations catalyzed by CYP2C9 and CYP2C8 and approximately 20 microM for activities catalyzed by CYP2C19 and CYP3A4 respectively. For the three metabolites of troglitazone tested, a quinone-type metabolite (M3) was the most potent inhibitor for CYP2C enzymes, followed by a sulphate conjugate (M1); effects of a glucuronide (M2) were very weak. The inhibitory effects of the parent drug were more potent than those of metabolites. Troglitazone and M3 inhibited P450 activities mainly through a competitive manner with Ki = 0.2-1.7 microM and 1.4-8.8 microM respectively. 4. In three human liver microsomes, troglitazone and its metabolites also inhibited paclitaxel 6alpha-hydroxylation, S-warfarin 7-hydroxylation, S-mephenytoin 4'-hydroxylation, and testosterone 6beta-hydroxylation with similar IC50, as observed for the recombinant P450 enzyme systems. 5. These results suggest that xenobiotic oxidations by P450 enzymes are more substantially affected by troglitazone and its metabolites than pioglitazone or rosiglitazone, and that drug interactions may be of much importance to understand the basis for the pharmacological and toxicological actions of this new oral antidiabetic drug.     

Cytochrome P450 inhibition using recombinant proteins and mass spectrometry/multiple reaction monitoring technology in a cassette incubation.

Detailed cytochrome P450 (P450) inhibition profiles are now required for the registration of novel molecular entities. This method uses combined substrates (phenacetin, diclofenac, S-mephenytoin, bufuralol, and midazolam) with combined recombinant P450 enzymes (CYP1A2, 2C9, 2C19, 2D6, and 3A4) in an attempt to limit interactions with other more minor P450s and associated reductases. Kinetic analysis of single substrate with single P450 (sP450) yielded apparent Km values of 25, 2, 20, 9, and 3 microM, for CYP1A2, 2C9, 2C19, 2D6, and 3A4, respectively. Combined substrates with combined P450s (cP450) yielded apparent Km values of 65, 4, 19, 7, and 2 microM. Selectivity of the substrates for each P450 isoform was checked. Phenacetin proved to be the least selective substrate. However, the ratio of the various P450s was modified in the final assay such that metabolism of phenacetin by other enzymes was approximately 20% of the metabolism by CYP1A2. IC50 determinations with alpha-naphthoflavone (0.04 microM), sulfaphenazole (0.26 microM), tranylcypromine (9 microM), quinidine (0.02 microM), and ketoconazole (0.01 microM) were similar for sP450 and cP450 enzymes. The assay was further evaluated with 11 literature compounds and 52 in-house new chemical entities, and the data compared with radiometric/fluorescent values. The overall protein level of the assay was reduced from the original starting point, as this led to some artificially high IC50 measurements when compared with existing lower protein assays (radiometric/fluorometric). This method offers high throughput P450 inhibition profiling with potential advantages over current radiometric or fluorometric methods.     

Identification of human cytochrome P450 and flavin-containing monooxygenase enzymes involved in the metabolism of lorcaserin, a novel selective human 5-hydroxytryptamine 2C agonist

Lorcaserin, a selective serotonin 5-hydroxytryptamine 2C receptor agonist, is being developed for weight management. The oxidative metabolism of lorcaserin, mediated by recombinant human cytochrome P450 (P450) and flavin-containing monooxygenase (FMO) enzymes, was examined in vitro to identify the enzymes involved in the generation of its primary oxidative metabolites, N-hydroxylorcaserin, 7-hydroxylorcaserin, 5-hydroxylorcaserin, and 1-hydroxylorcaserin. Human CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, CYP3A4, and FMO1 are major enzymes involved in N-hydroxylorcaserin; CYP2D6 and CYP3A4 are enzymes involved in 7-hydroxylorcaserin; CYP1A1, CYP1A2, CYP2D6, and CYP3A4 are enzymes involved in 5-hydroxylorcaserin; and CYP3A4 is an enzyme involved in 1-hydroxylorcaserin formation. In 16 individual human liver microsomal preparations (HLM), formation of N-hydroxylorcaserin was correlated with CYP2B6, 7-hydroxylorcaserin was correlated with CYP2D6, 5-hydroxylorcaserin was correlated with CYP1A2 and CYP3A4, and 1-hydroxylorcaserin was correlated with CYP3A4 activity at 10.0 μM lorcaserin. No correlation was observed for N-hydroxylorcaserin with any P450 marker substrate activity at 1.0 μM lorcaserin. N-Hydroxylorcaserin formation was not inhibited by CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, and CYP3A4 inhibitors at the highest concentration tested. Furafylline, quinidine, and ketoconazole, selective inhibitors of CYP1A2, CYP2D6, and CYP3A4, respectively, inhibited 5-hydroxylorcaserin (IC(50) = 1.914 μM), 7-hydroxylorcaserin (IC(50) = 0.213 μM), and 1-hydroxylorcaserin formation (IC(50) = 0.281 μM), respectively. N-Hydroxylorcaserin showed low and high K(m) components in HLM and 7-hydroxylorcaserin showed lower K(m) than 5-hydroxylorcaserin and 1-hydroxylorcaserin in HLM. The highest intrinsic clearance was observed for N-hydroxylorcaserin, followed by 7-hydroxylorcaserin, 5-hydroxylorcaserin, and 1-hydroxylorcaserin in HLM. Multiple human P450 and FMO enzymes catalyze the formation of four primary oxidative metabolites of lorcaserin, suggesting that lorcaserin has a low probability of drug-drug interactions by concomitant medications.     

Pathways of carbamazepine bioactivation in vitro I. Characterization of human cytochromes P450 responsible for the formation of 2- and 3-hydroxylated metabolites.

In vitro studies were conducted to identify the cytochromes P450 (P450s) involved in the formation of 2- and 3-hydroxycarbamazepine, metabolites that may serve as precursors in the formation of protein-reactive metabolites. Human liver microsomes (HLMs) converted carbamazepine (30-300 microM) to 3-hydroxycarbamazepine at rates >25 times those of 2-hydroxycarbamazepine. Both the 2- and 3-hydroxylation of carbamazepine appeared to conform to monophasic Michaelis-Menten kinetics in HLMs (apparent K(m) values, approximately 1640 and approximately 217 microM; apparent V(max) values, approximately 5.71 and approximately 46.9 pmol/mg of protein/min, respectively). Rates of carbamazepine 2- and 3-hydroxylation correlated strongly with CYP2B6 activity (r >or= 0.757) in a panel of HLMs (n = 8). Carbamazepine 3-hydroxylation also correlated significantly with CYP2C8 activity at a carbamazepine concentration of 30 microM. Formation of 2- and 3-hydroxycarbamazepine did not correlate significantly with any other P450 activities. The chemical inhibitors ketoconazole (CYP3A) and 7-EFC (CYP2B6) inhibited both 2- and 3-hydroxycarbamazepine formation whereas 4-methylpyrazole (CYP2E1) markedly decreased 2-hydroxycarbamazepine formation. Several recombinant P450s catalyzed carbamazepine 2- and 3-hydroxylation, but after adjustment for relative hepatic abundance, CYP3A4 and CYP2B6 appeared to be the major catalysts of carbamazepine 3-hydroxylase activity, and at least five P450s were significant contributors to 2-hydroxycarbamazepine formation; CYP2E1 made the greatest contribution to the Cl(int) of carbamazepine 2-hydroxylation (approximately 30%), but P450s CYP1A2, 2A6, 2B6, and 3A4 also made significant contributions (approximately 13-18%). These results suggest that CYP2B6 and CYP3A4 are largely responsible for the formation of 3-hyrdoxycarbamazepine, whereas multiple P450s (CYP1A2, 2A6, 2B6, 2E1, and 3A4) contributed to 2-hydroxycarbamazepine formation.     

Cytochrome P-450 3A and 2D6 catalyze ortho hydroxylation of 4-hydroxytamoxifen and 3-hydroxytamoxifen (droloxifene) yielding tamoxifen catechol: involvement of catechols in covalent binding to hepatic proteins.

Earlier study suggested that 3,4-dihydroxytamoxifen (tam catechol), a tamoxifen metabolite, is proximate to the reactive intermediate that binds covalently to proteins and possibly to DNA (). The current study demonstrates that rat and human hepatic cytochrome P-450s (CYPs) catalyze tam catechol formation from tamoxifen (tam), 3-hydroxy-tam (Droloxifene), and 4-hydroxy-tam (4-OH-tam). Higher levels of catechol were formed from 4-OH-tam and 3-hydroxy-tam than from tam. Evidence that human hepatic CYP3A4 and 2D6 catalyze the formation of tam catechol from 4-OH-tam and supportive data that the catechol is proximate to the reactive intermediate, was obtained: 1) There was a good correlation (r = 0.82; p 相似文献   

Characterization of human cytochrome p450 enzymes involved in the metabolism of cilostazol.

Cilostazol (OPC-13013; 6-[4-(1-cyclohexl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2(1H)-quinolinone) is widely used as an antiplatelet vasodilator agent. In vitro, the hydroxylation of the quinone moiety of cilostazol to OPC-13326 [6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-4-hydroxy-2(1H)-quinolinone], is the predominant route, and the hydroxylation of the hexane moiety to OPC-13217 is the second most predominant route. This study was carried out to identify and kinetically characterize the human cytochrome P450 (P450) isozymes responsible for the formation of the two major metabolites of cilostazol, namely, OPC-13326 and OPC-13217 [3,4-dihydro-6-[4-[1-(cis-4-hydroxycyclohexyl)-1H-tetrazol-5-yl)butoxy]-2(1H)-quinolinone)]. In in vitro studies using 14 recombinant human P450 isozymes, CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4, CYP3A5, and CYP4A11, cilostazol was metabolized to OPC-13326 mainly by CYP3A4 (K(m) = 5.26 muM, intrinsic clearance (CL(int)) = 0.34 microl/pmol P450/min), CYP1B1 (K(m) = 11.2 microM, CL(int) = 0.03 microl/pmol P450/min), and CYP3A5 (K(m) = 2.89 microM, CL(int) = 0.05 microl/pmol P450/min) and to OPC-13217 mainly by CYP3A5 (K(m) = 1.60 microM, CL(int) = 0.57 microl/pmol P450/min), CYP2C19 (K(m) = 5.95 microM, CL(int) = 0.16 microl/pmol P450/min), CYP3A4 (K(m) = 5.35 microM, CL(int) = 0.10 microl/pmol P450/min), and CYP2C8 (K(m) = 33.8 microM, CL(int) = 0.009 microl/pmol P450/min). The present study showed that the two major metabolites of cilostazol in vitro, namely, OPC-13326 and OPC-13217, are mainly catalyzed by CYP3A4 and CYP3A5, respectively.     

Involvement of human cytochrome P450 2B6 in the omega- and 4-hydroxylation of the anesthetic agent propofol

Human liver microsomal cytochrome P450s (P450s or CYP) involved in the oxidative biotransformation of the anesthetic agent propofol were investigated. Of six cDNA-expressed human P450 enzymes tested, CYP2B6 and CYP1A2, followed by CYP3A4, had high catalytic activities at a 20 microM propofol concentration, corresponding to clinical plasma levels. K(m) and k(cat) values for propofol omega- and 4-hydroxyation were 27 microM and 21 nmol omega-hydroxypropofol formed/min/nmol CYP2B6 and 30 microM and 42 nmol 4-hydroxypropofol formed/min/nmol CYP2B6, respectively. CYP2B6 expressed in HepG2 cells also effectively catalyzed propofol omega- and 4-hydroxylation. In a panel of individual human liver microsomes, propofol omega- and 4-hydroxylation activities (at the substrate concentration of 20 microM) were highly correlated with CYP2B6 contents, and moderately with CYP3A4 contents. Anti-CYP2B6 antibody inhibited both omega- and 4-hydroxylation activities in human liver samples that contained relatively high levels of CYP2B6, whereas alpha-naphthoflavone and an anti-CYP1A2 antibody showed inhibitory effects on the 4-hydroxylation activity in a liver microsomal sample in which the CYP1A2 level was relatively high. These results suggest that CYP2B6 has an important role in propofol omega- and 4-hydroxylation in human livers and that the hepatic contents of CYP2B6, CYP3A4, and CYP1A2 determine which P450 enzymes play major roles in propofol oxidation in individual humans.     

Identification of human cytochrome P450s involved in the formation of all-trans-retinoic acid principal metabolites

Cytochrome P450 (P450)-dependent metabolism of all-trans-retinoic acid (atRA) is important for the expression of its biological activity. Because the human P450s involved in the formation of the principal atRA metabolites have been only partially identified, the purpose of this study was to identify the human P450s involved in atRA metabolism. The use of phenotyped human liver microsomes (n = 16) allowed the identification of the following P450s: 2B6, 2C8, 3A4/5, and 2A6 were involved in the formation of 4-OH-RA and 4-oxo-RA; 2B6, 2C8, and 2A6 correlated with the formation of 18-OH-RA; and 2A6, 2B6, and 3A4/5 activities correlated with 5, 6-epoxy-RA formation (30-min incubation, 10 microM atRA, HPLC separation, UV detection 340 nm). The use of 15 cDNA-expressed human P450s from lymphoblast microsomes, showed the formation of 4-OH-RA by CYP3A7 > CYP3A5 > CYP2C18 > CYP2C8 > CYP3A4 > CYP2C9, whereas the 18-OH-RA formation involved CYPs 4A11 > 3A7 > 1A1 > 2C9 > 2C8 > 3A5 > 3A4 >2C18. Kinetic studies identified 3A7 as the most active P450 in the formation of three of the metabolites: for 4-OH-retinoic acid, 3A7 showed a V(max)/K(m) of 127.7, followed by 3A5 (V(max)/K(m) = 25.6), 2C8 (V(max)/K(m) = 24.5), 2C18 (V(max)/K(m) = 15.8), 3A4 (V(max)/K(m) = 5.7), 1A1 (V(max)/K(m) = 5.0), and 4A11 (V(max)/K(m) = 1.9); for 4-oxo-RA, 3A7 showed a V(max)/K(m) of 13.4, followed by a 10-fold lower activity for both 2C18 and 4A11 (V(max)/K(m) = 1.2); and for 18-OH-RA, 3A7 showed a V(max)/K(m) of 10.5 compared with a V(max)/K(m) of 2.1 for 4A11 and 2.0 for 2C8. 5,6-Epoxy-RA was only detected at high substrate concentrations in this system (>10 microM), and P450s 2C8, 2C9, and 1A1 were the most active in its formation. The use of embryonic kidney cells (293) stably transfected with human P450 cDNA confirmed the major involvement of P450s 3A7, 1A1, and 2C8 in the oxidation of atRA, and to a lesser extent, 1A2, 2C9, and 3A4. In conclusion, several human P450s involved in atRA metabolism have been identified, the expression of which was shown to direct atRA metabolism toward the formation of specific metabolites. The role of these human P450s in the biological and anticancer effects of atRA remains to be elucidated.